Titan Krios Cryo-EM – Access / Usage Policy
I. Introduction
The Titan Krios G3 Cryo-EM in the WUCCI is a state-of-the-art fully automated imaging robot designed for rapid, stable, high-throughput data collection on frozen-hydrated samples. It is an extremely expensive instrument, which is projected to be in demand by research groups at WUSTL and the surrounding region. To maximize access and sample throughput of this instrument, the WUCCI has put into place this policy for screening procedures and quality checks before any sample may be loaded into the Krios.
II. Access to the Titan Krios Cryo-EM
Step 1: Cryo-EM Project Request
The first step in requesting time on the Titan Krios is to complete and submit the Cryo-EM Project Request Form.
Step 2: Project Assessment
Following the project request submission, we will schedule a meeting to discuss project goals with the WUCCI Cryo-EM staff and verify project readiness. All students, post-doctoral fellows and research assistants/associates involved with the project are encouraged to attend this meeting. Attendance of the principal investigator is not required, but is recommended for a more effective assessment of the project. An estimated project cost will be calculated and provided as a quotation as a follow up to this meeting.
Step 3: Ad Hoc Committee Review
Once these two steps are complete, the submitted project will undergo review by the WUCCI Cryo-EM Ad Hoc Committee. This mechanism provides a confirmation of project appropriateness and will be used to establish priority for beam-time allocation. The ad hoc committee will be comprised of:
- WUCCI Scientific Director
- Titan Krios Manager
- 2 members of WUCCI advisory board
- 1 member of WUSTL structural biology community
and may not include any individual that has a project request in review in that current cycle. The ad hoc committee will meet every month, or as required to review applications.
Step 4: Project Approval
Once a project has been approved for imaging on the Titan Krios, beam-time will be allocated based on the priority ranking from the ad hoc committee review. Initially, a maximum of two (2) days of consecutive data acquisition will be allocated per PI per week and the number of slots allocated per PI per month will be capped at eight (8) days. Weekend use will be restricted to WUCCI cryo-EM staff and those users from cryo-EM centric labs that have been trained (see section IV).
Step 5: Sample Submission
Submission of samples will follow outlined procedures for cryo grid quality control as detailed below (see section III).
III. Procedure for Cryo Grid Quality Control
In order to maximize the success of imaging frozen samples on the Krios, it is first recommended that the researcher screen the quality of their sample by both negative-stain TEM and low-resolution cryo-EM.
Negative-stain is a conventional method whereby heavy metal salts such as uranyl acetate or uranyl formate give samples contrast and allow imaging on the WUCCI JEOL JEM-1400 120kV TEM. Cryo-EM screening will involve vitrification of the sample using the FEI Vitrobot Mk IV and imaging in the JEOL JEM-1400 120kV TEM using the Gatan 626 cryo-holder. Vitrification of samples will be provided for you by the Titan Krios Manager, Michael Rau. For samples to be approved for imaging on Titan Krios, the following quality control data must be submitted as a part of the Cryo-EM Project Request:
- Low magnification negative stain TEM images showing absence of aggregates and contamination in the sample.
- High-magnification negative stain TEM images showing particle distribution.
- Low magnification cryo-EM images showing high quality ice of appropriate thickness can be obtained for the sample in question.
- High-magnification cryo-EM images showing uniform particle distribution and absence of aggregation and contamination.
The most efficient way to ensure successful data collection on the Krios is to validate the Vitrobot blotting and concentration conditions by screening the grids with the JEOL JEM-1400 120kV TEM equipped with a Gatan 626 cryo-holder, and then freezing new grids using the optimized conditions. If screening one or two of the new grids shows that they have good ice and particle distribution, the rest of can be submitted for loading into the Krios. Following these steps will ensure the cryo grids have proper ice thickness, good particle distribution and sufficient usable area on a given grid, and thus, make efficient use of available Krios beam-time.
Users external to WUSTL who wish to ship their cryo grids to St. Louis should ensure they arrive within one-to-five days prior to the start of their session on the Krios. It is, however, acceptable for users to bring the grids with them, but they should be prepared to load the samples by 9:00 am on the first day of their scheduled beam-time allocation.
IV. Trained Use of the Titan Krios
The Titan Krios and its associated CMOS and Direct Electron detectors are specialized instruments that can be easily misaligned and even damaged by improper or negligent use. To maximize experimental throughput, the WUCCI will initially restrict the availability of trained user access to personnel who have had a minimum of 5 years experience using high-end cryo-electron microscopes (such as a 200kV Glacios ). This practice will facilitate safe operation of the instrument, minimize downtime, and maximize experimental throughput for the benefit of all investigators.
V. Titan Krios Alignments
The Krios is aligned daily by the Titan Krios Manager (Brock Summers). Users are not permitted to perform any alignments on the Krios in an unsupervised manner. Any user who misaligns the Krios will be charged the cost of realigning the microscope.
VI. Titan Krios Usage Charges
Usage on the Titan Krios will be allocated in daily slots. Hourly usage of the microscope (for either sample checking or tomographic acquisitions) will be billed at a rate of $100 per hr. Data acquisition runs will be billed per daily slot used. Recharge costs will be as follows:
- WUSTL
- Trained user – $1,760 per day
- Assisted user – $1,980 per day
- External Academic
- Assisted user – $2,970 per day
- Commercial
- Assisted user – $5,940 per day
If a sample turns out to be bad even after the screening procedures, or the data acquisition run fails, the run charges will be capped at 4 hrs @ screening rate. Those costs will be as follows:
- WUSTL – $400
- External Academic – $600
- Commercial – $1200
VII. Data Storage and Image Processing
Data generated on the Krios will be automatically deposited, via a dedicated 20Gbit/s fiber optic connection onto a local high-speed cache server in close proximity to the WUCCI. From there it will be automatically replicated using the IBM SpectrumScale GPFS parallel filesystem across the 40Gbit/s backbone of the Washington University Research Network (WURN) to the new Distributed Data Storage Network for Research Data. There the data will be organized and catalogued using the APPION cryo-EM data management pipeline, originally developed by the National Resource for Automated Molecular Microscopy (NRAMM). All costs associated with the storage of primary data will be covered in the Titan Krios usage fees. Users are at liberty to transfer their data to their own storage allocations, but should be aware that there may well be costs associated with that
Typically, the analysis of cryo-EM data will be the responsibility of each individual PI. The WUCCI has a small multi-GPU server running the RELION 2.0 software for 2D classification and 3D model refinement, and is happy to provide consultations on different processing approaches as well as assist users on a collaborative basis with 3D model building. In addition, structural biology expertise is available on an as needed basis from Prof. Andrzej Krezel, Department of Biochemistry and Molecular Biophysics.